PL206435B1 - Novel vaccine compositon - Google Patents

Description

2 PL 206 435 Β1

Description of the invention

The invention relates to an inactivated influenza virus preparation, an influenza vaccine, a method for producing a stable hemagglutinin antigen, the use of an influenza virus preparation, and the use of α-tocopherol succinate. In particular, the invention relates to vaccines containing inactivated influenza virus which are fragments rather than whole influenza vaccines and which are stable in the absence of organic mercury preservatives. Additionally, these vaccines contain haemagglutinin, which is stable according to known tests. These vaccines may be administered by any route suitable for the administration of such vaccines, such as intramuscular, subcutaneous, intradermal or mucosal, e.g. intranasal.

The influenza virus is one of the most widespread viruses in the world, affecting both humans and livestock. Flu has a significant impact on the economy.

The influenza virus is an RNA enveloped virus; its particle diameter is about 125 nm. It consists predominantly of an internal nucleocapsid or a nucleoprotein-bound ribonucleic acid (RNA) core surrounded by a viral envelope composed of a lipid bilayer and external glycoproteins. The inner layer of the viral envelope is mostly composed of matrix proteins and the outer layer is mainly of host-derived lipid material. The surface glycoproteins neuraminidase (NA) and haemagglutinin (HA) look like spikes, 10-12 nm long, on the surface of the molecule. These surface proteins, and in particular the haemagglutinin, determine the antigen specificity of the influenza virus subtypes.

Currently available vaccines contain either inactivated or live attenuated influenza virus. Inactivated influenza vaccines contain one of three possible forms of antigen preparation: whole inactivated virus, sub-virions in which the purified viral particles are fragmented with detergents or other reagents that dissolve the lipid envelope (so-called "split" vaccines) or purified HA and NA (subunit vaccines). Such inactivated vaccines are administered intramuscularly (i.m.) or intranasally (i.n.). Live attenuated vaccines are not commercially available.

All types of flu vaccines are usually trivalent vaccines. They mostly contain antigens derived from two strains of influenza A virus and one strain of influenza B. In most cases, a typical 0.5 ml injection dose contains 15 g of haemagglutinin antigen from each strain, as measured by single radial immunodiffusion (SRD) ) (JM Wood et al .: An improved single radial immuno-diffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determi-nation of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237- 247; JM Wood et al., International collaborative study of single radial diffusion and immunoelectrophoresis technique s for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981) 317-330). The World Health Organization, in collaboration with national health authorities and vaccine manufacturers, decides which flu strains will be included in the vaccine each season.

Typical influenza epidemics increase the incidence of pneumonia and lower respiratory disease, which is manifested by increased hospitalization and mortality rates. Such complications are more common in the elderly and those suffering from chronic diseases. Young children can also have severe flu. For this reason, these groups in particular need protection.

Current efforts to control the incidence and mortality associated with annual influenza epidemics rely on the intramuscular administration of vaccines containing inactivated influenza virus. The effectiveness of such vaccines in preventing respiratory disease and complications from influenza ranges from 75% in healthy adults to less than 50% in the elderly.

International standards are used to measure the effectiveness of influenza vaccines. The official European Union criteria for the effectiveness of the flu vaccine are shown in the table below. In theory, to meet European Union requirements, an influenza vaccine needs to meet only one of the criteria in the table for all strains of influenza included in the vaccine. In practice, however, it is necessary for all strains to meet at least two or even all three criteria, especially for new vaccines, such as new vaccines for administration by a different route. In some cases, meeting two 3 PL 206 435 wystarcz1 criteria may be sufficient. For example, it might be acceptable that two criteria would be met by all strains while the third criterion would be met by some but not all strains (e.g. two out of three strains). The requirements for the adult population (18-60 years old) and for the elderly population (> 60 years old) are different. 18-60 years > 60 years Seroconversion rate *> 40%> 30% Conversion rate " > 2,5 > 2,0 Protection percentage " * > 70% > 60%

The seroconversion rate is defined as the percentage of vaccinated subjects whose serum haemagglutinin (HI) inhibition titer increased at least 4-fold for each vaccine strain following vaccination. "The conversion rate is defined as the fold increase in plasma HI Geometric Mean Titer (GMT) after vaccination for each vaccine strain. "Protection rate is defined as the percentage of vaccinated subjects who have a plasma HI titer equal to or greater than 1:40 after vaccination (for each vaccine strain) and is normally considered the index of protection.

In order for a new influenza vaccine to be considered marketable, it must not only meet these standards, but must also in practice be at least as effective as currently available injectable vaccines. It must also be economically competitive in the amount of antigen required and the number of administrations required. Currently commercially available are either split or subunit injectable influenza vaccines. These vaccines are produced by fragmenting a viral particle, primarily using an organic solvent or detergent, and different degrees of separation or purification of viral proteins.

Split vaccines are prepared by fragmenting whole influenza virus, either infectious or inactivated, using dissolving concentrations of organic solvents or detergents, and then removing the solubilizing agent and some or most of the viral lipid material. Split vaccines generally contain contaminants in the form of matrix proteins and nucleoproteins, and sometimes lipids, as well as envelope membrane proteins. Split vaccines will usually contain most or all of the viral structural proteins, but not necessarily in the same proportions as are present throughout the virus. On the other hand, subunit vaccines contain essentially highly purified viral surface proteins, haemagglutinin and neuraminidase, which are surface proteins responsible for stimulating the production of the desired virus neutralizing antibodies upon vaccination.

Many vaccines today require the addition of a preservative to prevent vaccine spoilage. A frequently used preservative is thiomersal, which is a mercury-containing compound. Some public concerns have been expressed about the effects of mercury-containing compounds. There is no control system in place to detect the effects of low to medium doses of organic mercury compounds on nervous system development, and special studies of children who have received high doses of organic mercury compounds will be completed in a few years. Some commentators emphasize that the potential dangers of using thimerosal-containing vaccines (Offit; P.A. JAMA vol. 283; no. 16) should not be overstated. However, it would undoubtedly be beneficial to find alternative methods of making vaccines, replacing the use of thiomersal in the production process. Thus, there is a need to develop thimerosal-free vaccines, in particular vaccines such as influenza vaccines, the use of which is recommended annually, at least for certain patient populations.

Until now, it has been standard practice to use preservatives during the manufacturing / purification process and / or in the final vaccine in commercially available inactivated influenza vaccines. A preservative is required to protect the vaccine from the growth of microorganisms during the various purification steps. In the case of egg-based influenza vaccines, thiomersal is usually added to the raw allantoic fluid, and may be added a second time during viral processing. Thus, at the end of the process, residual amounts of thiomersal will be present, which can be further increased to the desired concentration of preservative in the final vaccine, e.g., to a concentration of about 100 µg / ml. 4 PL 206 435 Β1

A stabilizing effect is a side effect of using thiomersal as a preservative in influenza vaccines. Thiomersal in commercially available influenza vaccines stabilizes the HA component of the vaccine, in particular, but not exclusively, the HA of the B strain of influenza virus. Some A strain haemagglutinins, e.g. H3, may also require stabilization. Therefore, while it may be desirable to consider removing thiomersal from influenza vaccines, or at least reducing the concentration of thiomersal in the final vaccine, there is a problem to overcome, as without thiomersal the HA will not be sufficiently stable. In the present invention it has been discovered that it is possible to stabilize HA in inactivated influenza virus preparations using alternative reagents not containing organic mercury compounds. HA remains stable, meaning that it is detected over time by known quantitative methods, in particular SRD, to a greater extent than unstabilized antigen preparation produced using the same method but without the use of a stabilizing excipient. The SRD method is carried out as described above. Importantly, HA remains stable for up to 12 months, which is the general requirement for a final influenza vaccine.

Thus, the invention relates to an inactivated influenza virus preparation containing a haemagglutinin antigen stabilized without the use of thiomersal or at low levels of thiomersal, the haemagglutinin being detected in the SRD assay, which is characterized by containing sufficient α-tocopherol succinate to stabilize the haemagglutinin.

Preferably, the inactivated influenza virus preparation according to the invention contains α-tocopherol succinate in a concentration of 1 µg / ml -10 mg / ml, more preferably in a concentration of 10-500 µg / ml.

Moreover, preferably with respect to the inactivated influenza virus preparation according to the invention, the influenza virus antigen preparation is selected from the group consisting of split virus antigen preparations, subunit antigens and chemically inactivated or heat inactivated whole virus or inactivated by ultraviolet or heat radiation, more preferably the influenza antigen preparation is a split antigen and virus preparation.

It is furthermore preferred that the inactivated influenza virus preparation according to the invention comprises both A and B strain hema-glutinin.

More preferably, the inactivated influenza virus preparation according to the invention is a trivalent influenza virus preparation.

Furthermore, it is preferred that the inactivated influenza virus preparation according to the invention comprises stabilized hemagglutinin of the influenza B strain.

Furthermore, the invention relates to an influenza vaccine which is characterized in that it comprises an influenza virus preparation as defined above.

Preferably, in the influenza vaccine according to the invention, the concentration of the haemagglutinin antigen for each of the influenza strains is between 1 and 1000 µg / ml, as determined by the SRD test.

Preferably, the influenza vaccine according to the invention additionally comprises an adjuvant.

Furthermore, the invention relates to a method for producing a stable haemagglutinin antigen, which is characterized in that the antigen is purified in the presence of α-tocopherol or a derivative thereof, such as α-tocopherol succinate.

Furthermore, the invention relates to the use of an inactivated influenza virus preparation for the production of a vaccine as defined above for the prophylaxis of influenza infection or illness in a subject.

Preferably with regard to the above use, the vaccine is administered by intradermal, intranasal, intramuscular, oral or subcutaneous routes.

In addition, the invention relates to the use of α-tocopherol succinate as a haemagglutinin stabilizer for the preparation of an influenza vaccine.

The low levels of thiomersal are those levels where the stability of the influenza B derived HA is reduced and a stabilizing excipient is required to stabilize the HA. Low levels of thiomersal are usually µg / ml or less.

Generally, stabilized HA refers to HA that is detectable over time by known quantitative methods, in particular SRD, to a greater extent than the unstabilized antigen preparation produced using the same method but without the use of a stabilizing excipient. Stabilization of HA advantageously allows the activity of HA to be kept substantially constant over a period of one year. Preferably, the stabilization allows the HA containing vaccine to provide acceptable protection after a 6 month storage period, more preferably after a 1 year period.

Suitably, stabilization is performed with a stabilizing excipient, preferably a micelle modifying excipient. A micelle modifying excipient is generally an excipient that can be incorporated into micelles formed by detergents used or suitable for solubilizing the membrane HA protein, such as the Tween 80, Triton Χ100 and deoxycholate detergents used individually or in combination.

Without wishing to be bound by theory, it is believed that excipients stabilize HA by interacting with lipids, detergents, and / or proteins in the final formulation. Mixed excipient micelles with proteins and lipids may be formed, such as Tween and deoxycholate micelles with residual lipids and / or Tritron Χ100. The surface proteins are believed to be kept solubilized by these complex micelles. Preferably, protein aggregation is limited by the repulsion of the charges of micelles containing suitable excipients, such as micelles containing negatively charged detergents.

Examples of suitable micelle modifying excipients are: positively, negatively, or zwitterionically charged amphiphilic molecules such as alkyl sulfates or alkylaryl sulfates; non-ionic amphiphilic molecules such as alkylpolyglycosides or their derivatives such as Plantacare® (available from Henkel KGaA) or polyalkylene alkyl alcohol ethers or derivatives thereof such as Laureth-9.

The preferred excipients are α-tocopherol or α-tocopherol derivatives, such as α-tocopherol succinate. Other exemplary tocopherol derivatives are D-α tocopherol, D-δ toco-ferol, D-γ tocopherol and DL-α tocopherol. Suitable tocopherol derivatives that can be used include acetates, succinates, phosphoric acid esters, formates, propionates, butyrates, sulfates, and gluconates. Α-Tocopherol succinate is used in the present invention. The α-tocopherol or a derivative thereof is present in an amount sufficient to stabilize the haemagglutinin.

Other suitable excipients can be identified by known methods and tested, e.g. in the SRD stability assay as described herein. In a preferred aspect, the invention provides an influenza virus antigen preparation comprising at least one stable influenza B strain hemagglutinin antigen.

The antigen preparation vaccines described herein find use in a method of preventing influenza infection or illness in a subject, the method comprising administering to that individual a vaccine according to the invention.

The vaccine may be administered by any suitable route, that is intradermally, intramuscularly, e.g. intranasally, orally, intramuscularly or subcutaneously. Other routes of administration are well known.

Intradermal administration is preferred. Any suitable device may be used for intradermal administration, e.g. a short needle device such as those described in the United States patents: US 4,886,499, US 5,190,521, US 5,328,483, US 5,527,288, US 4,270,537, US 5,015,235, US 5,141,496, US 5,417662. Vaccines can also be administered intradermally using devices that limit the extent of effective needle penetration into the skin, such as those described in WO 99/34850 and EP 1092444, and their equivalent in performance. Devices such as a jet injector are also useful. They deliver liquid vaccines to the dermis using a needle-free syringe to deliver the fluid or with a needle that penetrates the stratum corneum and produces a stream that enters the dermis. Needle-free syringes are described e.g. in the United States patents: US 5,480,381, US 5,599,302, US 5,334,144, US 5,993,412, US 5,649,912, US 5,569,189, US 5,704,911, US 5,383,851, US 5,893,397, US 5,466,220, US 5,339,163, US 5,312,335, US 5,503,627, US 5,064,413, US 5,520,639, US 4,596,556, US 4,790,824, US 4,941,880, US 4,940,460, WO 97/37705 and WO 97/13537. Ballistic powder / particle delivery devices that use pressurized gas to accelerate the migration of the powder vaccine through the outer layers of the skin to the dermis are also useful. Additionally, conventional syringes can be used for classical intradermal administration by the mantoux method. However, the usual use of syringes requires highly skilled personnel, so devices which allow accurate administration not only by highly skilled personnel are preferred. 6 PL 206 435 Β1

The invention finds use in a method for the prophylaxis of influenza infection or illness in a subject, according to which the subject is administered an intradermal influenza vaccine according to the invention.

The invention also includes devices for intradermal administration in combination with a vaccine of the invention, in particular, e.g., the devices disclosed in WO 99/34850 or EP 1092444.

The invention is applicable in particular, but not exclusively, to the stabilization of the hemagglutinin of influenza B strain.

Preferably, the stabilized HA of the invention is stable for a period of 6 months, more preferably for a period of 12 months. α-Tocopherol is in the form of succinate.

The preferred concentration of α-tocopherol or derivative is 1 µg / ml -10 mg / ml, more preferably 10-500 µg / ml.

The vaccine of the invention generally comprises both A and B strain antigens of influenza virus, typically in a trivalent composition of two A strains and one B strain. However, bivalent and monovalent vaccines are not excluded. Monovalent vaccines may be of benefit in, for example, a pandemic where it is important to produce and administer a vaccine as soon as possible.

Non-viable influenza antigen preparations suitable for use in the invention can be selected from the group consisting of split virus antigens preparations, subunit antigens (both recombinantly expressed and whole viral particle derived), whole virus inactivated, which can be chemically inactivated by using e.g. formaldehyde, β-propiolactone, or otherwise inactivated, e.g. by ultraviolet radiation or heat. Preferably the antigen preparation is a split virus preparation or a subunit preparation obtained from whole virus, in particular by cleavage and subsequent purification of the surface antigen. Split virus preparations are most preferred.

Preferably, the concentration of the haemagglutinin antigen for a preparation of each influenza virus strain, as determined by the SRD assay, is 1-000 µg / ml, more preferably 3-300 µg / ml and most preferably about 30 µg / ml.

The vaccine of the invention may additionally include an adjuvant or immunostimulator such as, but not limited to, detoxified lipid A derived from any source or non-toxic lipid A derivatives, saponins, and other reagents capable of stimulating a TH1-type response.

It has long been known that enterobacterial lipopolysaccharide (LPS) is a potent stimulator of the immune system, although its use as adjuvants is limited by its toxic effects. The non-toxic derivative of LPS, monophosphoryl lipid A (MPL) obtained by removing the carbohydrate group of the core and the phosphate from the reducing end of glucosamine, is described by Ribi et al (1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, pp. 407-) 419), and has the following formula:

HO

7 PL 206 435 Β1

Another detoxified variant of MPL is obtained by removing the acyl chain from position 3 of the disaccharide backbone, and is referred to as 3-O-deacylated monophosphoryl lipid A (3D-MPL). It can be purified and prepared by the methods described in GB 2122204B, which also relates to the preparation of diphosphoryl lipid A and its 3-O-deacylated variants.

A preferred form of 3D-MPL is in the form of a small particle emulsion less than 0.2 μίτι in diameter. A method for its production is disclosed in WO 94/21292. Aqueous formulations containing monophosphoryl lipid A and a surfactant are described in WO 9843670 A2.

The adjuvants derived from bacterial lipopolysaccharide to be formulated in the compositions of the invention may be adjuvants purified and processed from bacterial sources, or alternatively the adjuvants may be synthetic. For example, purified monophosphoryl lipid A has been described by Ribi et al. 1986 (see above), 3-O-deacylated monophosphoryl- or diphosphoryl lipid A obtained from Salmonella sp. Is described in GB 2,220,211 and US 4,912,094. Other purified and synthetic lipopolysaccharides are also described (Hilgers et al., 1986, Int. Arch. Allergy. Immunol., 79 (4): 392-6; Hilgers et al., 1987, Immunology, 60 (1): 141-6 and EP 0549074 B1). A particularly preferred bacterial lipopolysaccharide adjuvant is 3D-MPL. Accordingly, the LPS derivatives that can be used according to the invention are those immunostimulants that are similar in structure to LPS, MPL or 3D-MPL. In a further aspect of the invention, the LPS derivatives may be acylated monosaccharides that form part of the MPL structure as described above.

Saponins are described in Lacaille-Dubois M. and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp. 363-386). Saponins are steroidal or triterpene glycosides that are widely distributed in the plant and marine animal world. Saponins are known to form colloidal solutions in water, which foam when shaken, and to precipitate cholesterol. When saponins are close to the cell membrane, they create pore-like structures in the cell membrane, which cause the cell membrane to rupture. An example of this is erythrocyte hemolysis, which is a property of some, but not all, saponins.

Saponins are used as adjuvants in vaccines intended for systemic administration. The adjuvant activity and haemolytic activity of individual saponins has been extensively studied in the professional literature (Lacaille-Dubios and Wagner, see above). For example, Quil A (derived from the bark of the South American tree Ouillaja Saponaria Molina) and its fractions are described in US 5,057,540 and in "Saponins as vaccine adjuvants ", Kensil CR, Crił Rev Ther Drug Carrier Sysł, 1996, 12 (1-2): 1 -55; and in EP 0362279 B1. Particulate structures, referred to as Immunostimulating Complexes (ISCOMS), containing Quil A fractions, have a haemolytic effect and have been used in the production of vaccines (Morein B., EP 0109942 B1; WO 96/11711; WO 96/33739). Hemolytic saponins QS21 and QS17 (HPLC-purified Quil A fractions) and are described as potent systemic adjuvants, and a method for their preparation is disclosed in US Patent No. 5,057,540 and EP 0362279 B1. Other saponins that have been used in vaccine studies for systemic administration include saponins from other plant species such as Gypsophilia and Saponaria (Bomford et al., Vaccine, 10 (9): 572-577, 1992).

The enhancement system comprises a combination of a non-toxic lipid A derivative and a saponin derivative, especially a combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic combination in which QS21 is suppressed by cholesterol as disclosed in WO 96/33739.

A particularly potent adjuvant composition is the formulation comprising QS21 and 3D-MPL in the oil in water emulsion described in WO 95/17210 and is a preferred composition.

Thus, in one embodiment, the invention provides a vaccine comprising the influenza antigen preparation of the invention with an adjuvant that is detoxified lipid A or a non-toxic derivative of lipid A, more preferably an adjuvant that is monophosphoryl lipid A or a derivative thereof.

Preferably, the vaccine additionally comprises a saponin, more preferably QS21.

Preferably the composition additionally comprises an oil-in-water emulsion. The vaccine composition is prepared by mixing the antigen preparation of the invention with a pharmaceutically acceptable excipient such as 3D-MPL.

The vaccines according to the invention may furthermore contain at least one surfactant, which may in particular be a nonionic surfactant. A suitable nonionic surfactant is selected from the group consisting of octyl- or nonylphenoxypolyoxyethanols (e.g. the commercially available Triton ™ series), polyoxyethylene sorbitan esters (Tween ™ series), and polyoxyethylene ethers or esters of general formula (I): 206 435 Β1

(I) H0 (CH2CH2O) n-A-R where n is 1-50, A is a bond or -C (O) -, R is C1-10 alkyl or phenyl-C1-10-alkyl; or a combination of two or more of the above measures.

The preferred surfactants of formula (I) are molecules wherein n is 4-24, more preferably 6-12, most preferably 9; the R component is C 1-20, more preferably C 4 -C 20 alkyl, and most preferably C 12 alkyl.

Octylphenoxypolyoxyethanols and polyoxyethylene sorbitan esters are described in "Surfactant systems" edited by Attwood and Florence (1983, Chapman and Hall). Octylphenoxypolyoxyethanols (octoxynols), including t-octylphenoxypolyethoxyethanol (Triton®-100 ™) are also described in Merck Index, entry 6858 (page 1162, 12th edition, Merck & Co. Inc., Whitehouse Station, NJ, USA; ISBN 0911910 -12-3). Polyoxyethylene sorbitan esters, including polyoxyethylene sorbitan monooleate (Tween 80 ™) are described in Merck Index, entry 7742 (page 1308, 12th edition, Merck & Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Both can be prepared by the method described therein, or purchased from commercial sources such as Sigma Inc.

Particularly preferred nonionic surfactants include Triton Χ-45, t-octylphenoxypolyethoxyethanol (Triton Χ-100), Triton Χ-102, Triton Χ-114, Triton Χ-165, Triton Χ-205, Triton Χ-305, Triton N -57, Triton N-101, Triton N-128, Breij 35, polyoxyethylene-9-lauryl ether (laureth 9) and polyoxyethylene-9-stearyl ether (steareth 9). Triton-100 and laureth 9 are particularly preferred. Polyoxyethylene sorbitan ester, polyoxyethylene sorbitan monooleate (Tween 80 ™) is also particularly preferred.

Further suitable polyoxyethylene ethers of general formula (I) are selected from the following group: polyoxyethylene 8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.

Alternative terms or names for polyoxyethylene lauryl ether are contained in the CAS Registry. The CAS registry number for polyoxyethylene-9-lauryl ether is 9002-92-0. Ethers and polyoxyethylene such as polyoxyethylene lauryl ether are described in Merck Index (12th Ed .: entry 7717, Merck & Co. Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Laureth 9 is produced by reacting ethylene oxide with dodecyl alcohol and has an average of nine ethylene oxide units. Two or more nonionic surfactants from the various groups of surfactants described may be present in the vaccine formulation as described. In particular, a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80 ™) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton Χ-100 ™) is particularly preferred. Another particularly preferred combination of nonionic surfactants includes laureth 9 and polyoxyethylene sorbitan ester and / or octoxynol.

Nonionic surfactants such as those discussed above are preferably present in the final vaccine composition at the following concentrations: polyoxyethylene sorbitan esters, including Tween 80 ™: 0.01 -1%, most preferably about 0.1% (w / v) ; octyl- or nonyl-phenoxypolyoxyethanols such as Triton®-100 ™ or other Triton detergents: 0.001-0.1%, most preferably 0.005-0.02% (w / v); polyoxyethylene ethers of general formula (I), such as laureth 9: 0.1-20%, preferably 0.1-10% and most preferably 0.1-1% or about 0.5% (w / v) . With certain vaccine formulations, other vaccine components may be included in the formulation. Thus, the compositions according to the invention may also contain bile acids or their derivatives, in particular in the form of a salt. These include the cholic acid derivatives and their salts, in particular the sodium salts of cholic acid or cholic acid derivatives. Examples of bile acids and their derivatives include cholic acid, deoxycholic acid, chenodesoxycholic acid, lithocholic acid, ursodeoxycholic acid, hiodesoxycholic acid, and derivatives such as glyco-, tauro-, amidopropyl-1-propanesulfonic acid, amidopropyl-2-hydroxy -1-propansulfonic acid derivatives of the above-mentioned bile acids or N, N-bis (3-D-gluconamidopropyl) deoxycholamide. A particularly preferred example is sodium deoxycholate (Na-DOC), which may be present in the final dose of the vaccine.

The invention also provides pharmaceutical kits comprising a vaccine delivery device filled with a vaccine according to the invention. Such delivery devices include, but are not limited to, needle devices, fluid delivery devices, needleless devices, powder devices, and aerosol devices (for nasal administration). 9 PL 206 435 Β1

The influenza virus antigen preparations of the present invention may be obtained by the usual embryonated egg method, or may be obtained by any new generation process that uses tissue culture to multiply the virus or express recombinant influenza virus surface antigens. Suitable cell substrates for virus multiplication include, e.g., canine kidney cells such as MDCK or cells from MDCK clones, MDCK-like cells, monkey kidney cells such as AGMK, which include Vero cells, suitable porcine cell lines or any other type. mammalian cells that are suitable for producing influenza virus for vaccine purposes. Suitable cell substrates also include human cells, e.g. MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example, primary cells such as chicken embryo fibroblasts are also included.

The influenza virus antigen preparation can be produced by any suitable commercially available process, e.g., the influenza virus cleavage process described in DD 300 833 and DD 211 444. Traditionally, split influenza virus has been produced using solvents / detergents. such as tri-n-butyl phosphate or diethyl ether in combination with Twe-en ™ (a process known as "Tween-ether" cleavage). This process is still in use at some production sites. Other disintegrants currently used include detergents or proteolytic enzymes or bile salts, e.g. sodium deoxycholate, as described in DD 155 875. Detergents that can be used as disintegrants include cationic detergents, e.g. cetyltrimethylammonium bromide (CTAB), other ionic detergents e.g. lauryl sulfate, taurodeoxycholate or non-ionic detergents such as those described above which include Triton Χ-100 (e.g. in the method described by Lin et al, 2000, Biologicals 28, 95-103 ) and Triton N-101, or a combination of any two or more detergents.

The method of producing a split vaccine will include a plurality of different filtration steps and / or other separation steps such as ultracentrifugation, ultrafiltration, zone centrifugation and chromatography (e.g. ion exchange), in various combinations, and optionally an inactivation step, e.g., heat, using formaldehyde or β-propiolactone or ultraviolet rays, which can be applied before or after cleavage. The splitting process may be batchwise, continuously or semi-continuously.

Preferred influenza antigen preparations in the split vaccine according to the invention contain residual amounts of Tween 80 and / or Triton Χ-100 from the manufacturing process, although these may also be added or their concentration adjusted after the split antigen is produced. Preferably, both Tween 80 and Triton Χ-100 are present. The preferred final concentration ranges of these nonionic surfactants in a vaccine dose are:

Tween 80: 0.01-1%, more preferably about 0.1% (v / v)

Triton Χ-100: 0.001-0.1% (w / v), more preferably 0.005-0.02% (w / v).

Alternatively, the influenza virus antigen preparations of the invention may be derived from a source other than the live influenza virus, e.g., the haemagglutinin antigen may be recombinantly produced.

The invention will now be described further in the following non-limiting examples.

Examples

Example 1. Preparation of an influenza virus antigen preparation using α-tocopherol succinate as a stabilizer in a vaccine without preservatives (with reduced thiomersal content).

A monovalent split vaccine was prepared according to the following procedure.

Preparation of viral inoculum On the day of inoculation of embryo-containing eggs, fresh inoculum is prepared by mixing the working inoculum with phosphate buffered saline containing gentamicin sulphate at 0.5 mg / ml and hydrocortisone at a concentration of 25 μg / ml. (depending on virus strain). Virus inoculum is stored at 2-8 ° C.

Inoculation of eggs containing embryos

9-11 day old embryonated eggs are used for virus replication. The shells are disinfected. The eggs are inoculated with 0.2 ml of viral inoculum material. The inoculated eggs are incubated at an appropriate temperature (depending on the virus strain) for 48 - 96 hours. At the end of the incubation period, the embryos are killed by chilling and the eggs are stored for 12-60 hours at 2-8 ° C. 10 PL 206 435 Β1

Collecting

Allantoic fluid from chilled embryonated eggs is collected. Typically 8-10 ml of raw allantoic fluid is obtained from one egg.

Concentration and isolation of whole virus particles from the allantoic fluid 1. Clarification of the fluid

The obtained allantoic fluid is clarified by centrifugation at moderate speed (in the range of 4000 - 14000 g). 2. The adsorption stage

To obtain a CaHP04 gel in the clear virus harvest, 0.5 mol / L Na2HP04 and 0.5 mol / L CaCl2 solution are added so as to obtain a final CaHP04 concentration of 1.5 g - 3.5 g CaHP04 / L depending on from the virus strain.

After sedimentation for at least 8 hours, the supernatant is removed and the influenza virus pellet is resolubilized by adding a 0.26 mol / L EDTA-Na2 solution depending on the amount of CaHP04 used. 3. Filtration

The resuspended precipitate is filtered through a 6 μηι filter membrane. 4. Sucrose gradient centrifugation

The influenza virus is concentrated by isopic centrifugation on a linear sucrose gradient (0.55% (w / v)) containing 100 µg / ml Thiomersal. The flow rate is 8-15 l / hour.

At the end of centrifugation, four different fractions are recovered from the rotor (sucrose is measured in a refractometer): - fraction 1 55-52% sucrose - fraction 2 about 52-38% sucrose - fraction 3 38-20% sucrose * - fraction 4 20-0 % sucrose * depending on the virus strain: fraction 3 can be reduced to 15% sucrose.

Only fractions 2 and 3 are used for the further production of the vaccine.

Fraction 3 is washed in the diafiltration process with phosphate buffer to reduce the sucrose content to less than about 6%. The influenza virus present in this dilution is centrifuged to remove soluble contaminants.

The sediment is resuspended and thoroughly mixed to obtain a homogeneous suspension. Fraction 2 and the resuspended fraction 3 are combined and phosphate buffer is added to obtain a volume of approximately 40 liters. This product is the monovalent whole virus concentrate. 5. Sucrose gradient centrifugation with sodium deoxycholate

The monovalent whole influenza virus concentrate is loaded into an ENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrose gradient (0.55% (w / v)) over which an additional sodium deoxycholate gradient is overlaid. Tween 80 is present at a concentration of up to 0.1 (w / v) during the splitting and tocopherol succinate is added in concentrations of up to 0.5 mM for B-strain viruses. The maximum sodium deoxycholate concentration is 0.7-1.5% (w / v) and is strain dependent. The flow rate is 8-15 liters / hour.

After completion of the centrifugation, the rotor contents are recovered in three different fractions (the sucrose is measured in a refractometer). Fraction 2 is used for further processing. The sucrose limits (47-18%) vary from strain to strain and are set after evaluation. 6. Sterile filtration

The split virus fraction is filtered through filter membranes ending with a 0.2 μηι membrane. For dilution, a solution containing a phosphate buffer, Tween 80 at 0.025% (w / v) and (for B virus strains) tocopherol succinate at a concentration of 0.5 mM is used. The final volume of the filtered fraction is 2-5 times the original volume of the fraction. 7. Inactivation

The filtered monovalent material is incubated at 22 ± 2 ° C for a maximum of 84 hours (depending on the virus strain, the incubation time may be shortened). Phosphate buffer containing 0.025% (w / v) Tween 80 is then added to reduce the total protein content to a maximum of 250 µg / ml. For B-strain viruses, phosphate buffered saline containing 0.025% (w / v) Tween 80 and 0.25 mM tocopherol succinate is used to dilute the total protein content to 250 μg / ml. . Formaldehyde is added to a final concentration of 50 µg / ml and inactivation is carried out at 20 ± 2 ° C for at least 72 hours. 8. Ultrafiltration

The inactivated split viral material is concentrated at least 2 times in a ultrafiltration unit, equipped with cellulose acetate membranes with 20 kDa MWCO. The material is then washed with phosphate buffer containing 0.025% (w / v) Tween 80 followed by phosphate buffered saline containing 0.01% (w / v) Tween. For B-strain virus, phosphate buffered saline containing 0.01% (w / v) Tween 80 and 0.1 mM tocopherol succinate is used for washing. 9. Final sterile filtration

The ultrafiltrated material is filtered through membranes ending with a 0.2 μίτι membrane. The filtration membranes are rinsed and the material is diluted if necessary so that the protein concentration does not exceed 500 μg / ml with phosphate buffered saline containing 0.01% (w / v) Tween 80 and (for B strains of viruses) 0.1 mM tocopherol succinate. 10. Storage

The final monovalent batch is stored at 2-8 ° C for a maximum of 18 months.

Stability

Table 1. Comparison of changes in HA content (pg / ml) over time, detectable in the SRD assay in the final monovalent batches

Strain Stabilizator After production 4 weeks at 30 ° C 6 months at 2-8 ^ 12 months at 2-8 ° CB / Yamanashi / 166/98 Tocopherol succinate (residual mercury 3 pg / ml) 169 139 (82%) 172 (> 100%) NB B / Yamanashi / 166/98 Thiomersal (108 μg / ml) 192 160 (83%) 186 (97%) 178 (93%) B / Yamanashi / 166/98 None (residual mercury 3 pg / ml ) 191 122 (60%) 175 (92%) 154 (81%) B / Johannesburg / 5/99 Tocopherol succinate (residual mercury 4 pg / ml) 166 183 (> 100%) 158 (95%) 179 (> ; 100%) B / Johannesburg / 5/99 Tocopherol succinate (residual mercury 4 pg / ml) 167 179 (> 100%) 158 (95%) 178 (> 100%) B / Johannesburg / 5/99 Tocopherol succinate (residual mercury 3 pg / ml) 144 151 (> 100%) 130 (90%) 145 (> 100%) B / Johannesburg / 5/99 * Thiomersal 159 NB 172 (> 100%) 154 (97%) ) B / Johannesburg / 5/99 ** None 169 107 (63%) 153 (90%) WB * manufactured under license for FLUARIX ™, ** manufactured in accordance with example 1, without tocoterol succinate, WB: under research, NB: not tested.

Example 2. Preparation of an influenza vaccine using α-tocopherol succinate as stabilizer in a vaccine with reduced thiomersal content

The final monovalent lots of the three strains, A / New Caledonia / 20/99 (H1N1) IVR-116, A / Panama / 2007/99 (H3N2) Resvir-17, and B / Yamanashi / 166/98, were prepared as described in Example 1. Connection

Appropriate amounts of monovalent final lots were combined to achieve a final concentration of 30 μg / ml of A / New Caledonia / 20/99 (H1N1) IVR-116 and A / Panama- / 2007/99 (H3N2) Resvir-17 respectively, and a concentration of 39 μg / ml B / Yamanashi / 166/98. The concentrations of Tween 80 and Triton X-100 were set at 580 µg / ml and 90 µg / ml, respectively. The final volume was made up to 3 liters with phosphate buffered saline. The trivalent harvest was filtered through the set terminated with a cellulose acetate membrane 12 PL 206 435 Β1 0.8 μίτι and the final trivalent batch was obtained. The final trivalent lot was placed in syringes of at least 0.5 ml each.

Table 2. Comparison of changes in HA content over time, detected in the SRD assay, in the final trivalent lots that were obtained from the syringes

Vaccine preparation Strain Month 0 Month 2 Month 4 Month 6 Influenza vaccine without stabilizer A / NCal / 20/99 33 (32-34) 32 (31-33) 36 (34-38) 31 (30-32) A / Pan / 2007/99 29 (27-31) 31 (28-34) 34 (32-36) 32 (31-33) B / Yam / 166/98 36 (34-38) 33 (32-34) 32 (30 -34) 31 (29-33) Influenza vaccine containing a-tocopherol succinate A / NCal / 20/99 31 (30-32) 32 (31-33) 36 (34-38) 32 (31-33) A / Pan / 2007/99 33 (30-36) 33 (30-36) 36 (35-37) 33 (31 -35) B / Yam / 166/98 37 (35-39) 36 (34-38) 38 ( 35-41) 36 (33-39)

Example 3. SRD assay used to measure the haemagglutinin content Glass plates (12.4 - 10.0 cm) are coated with an agar gel containing the HA flu serum at the NIBSC recommended concentration. After the gel has set in the agar, 72 sample wells (3 mm 0) are punched out. 10 μΙ of the appropriately diluted standard and samples are placed in the wells. The plates are incubated for 24 hours at room temperature (20-25 ° C) in a humid chamber. The plates are then soaked overnight with NaCl solution and washed briefly with distilled water. The gel is then squeezed and dried. When the plates are completely dry, they are stained with Coomassie Brilliant Blue solution for 10 minutes and decolorized twice in a mixture of methanol and acetic acid until clearly marked stained areas appear. After the plates are dried, the diameter of the stained areas around the antigen wells is measured in the two directions crossing at right angles. Alternatively, an area measuring apparatus can be used. Dose-response curves of antigen dilutions versus area are constructed, and results are calculated using known slope-ratio methods (Finney DJ (1952). Statistical Methods in Biological Assay. London: Griffin, cited in Wood JM et al. (1977) J. Biol. Standard 5,237-247).

Example 4. Clinical studies of α-tocopherol stabilized influenza vaccine (thiomersal reduced)

The syringes prepared as described in example 2 are used in clinical trial H3N2: A / Panama / 2007/99 RESVIR-17 H1N1: A / New Caledonia / 2 0/99 (H1N1) IVR-116 B: B / Yamanashi / 166 / 98

Table 3

Adults aged 18-60 years Reduced-thio Plus-thio 1 2 3 4 5 6 7 8 H3N2 H1N1 B H3N2 H1N1 B pre-strain. GMT 47 41 111 55 37 102 Titer < 10 [%] 10.3% 13.8% 1.7% 5.3% 12.3% 8.8% Titre > 40, SPR [%] 60.3% 55.2% 75.9% 70.2% 52.6% 75.4% post-strain. Seroconversion rate [%] 10.3% 13.8% 1.7% 5.3% 12.3% 8.8% Significant increase in antibody titer [%] 58.6% 74.1% 58.6% 63, 2% 73.7% 52.6% PL 206 435 Β1 13 cont. table 3 1 2 3 4 5 6 7 8 Seroconversions [%] 58.6% 74.1% 58.6% 63.2% 73.7% 52.6% GMT 328 525 766 324 359 588 Multiplicity of GMT 7.3 13.0 6.9 5.9 9.8 5.9 Titre > 40, SPR [%] 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

Not performed = PU (confidence interval) for the proportion p = n / N was not determined because p * (1 -p) * N < 9 n / N = corresponding to (n) as a percentage of the number of individuals of the (sub) population (N) , i.e. seroconversions or significant increases, see also CPMAP / BWP / 214/96, March 12, 1997, p. 17 et seq. GMT = Geometric Mean Titer, Inverse 95% PU = 95% Confidence Interval

SPR = Rate of seroprotection: proportion of individuals with a protective titre before or after vaccination > Titer = HI antibody titer

Seroconversion rate = proportion of individuals with an increase in antibody titer from <10 pre-implantation to> 40 post vaccination GMT fold = fold increase in GMT

Significant increase = proportion of individuals with pre-vaccination antibody titre> 10 and a 4-fold post-vaccination antibody titer increase (two-step titer)

Dim. = EU requirements

Seroconversions = negative to positive or 4-fold increase (negative: titer &lt; 10, positive: titer &gt; 40) = proportion of individuals either seroconverted (&lt; 10 to &gt; 40) or with a significant increase.

The results show that the vaccine can provide the same protection as vaccines containing thiomersal as a preservative.

Example 5a. Influenza antigen preparation using α-tocopherol succinate as stabilizer in a thiomersal free vaccine

A monovalent split vaccine is prepared according to the following procedure.

Preparation of viral inoculum On the day of inoculation with embryo eggs, fresh inoculum is prepared by mixing the working inoculum with phosphate buffered saline containing gentamicin sulfate at a concentration of 0.5 mg / ml and hydrocortisone at a concentration of 25 µg / ml. (depending on virus strain). Virus inoculum is stored at 2-8¾.

Inoculation of eggs containing embryos

9-11 day old embryonated eggs are used for virus replication. The shells are sanitized. The eggs are inoculated with 0.2 ml of viral inoculum. 60,000 inoculated inoculated eggs are incubated at the appropriate temperature (depending on the virus strain) for 48-96 hours. At the end of the incubation period, the embryos are killed by chilling and the eggs are stored for 12-60 hours at 2-8 ° C.

Collecting

Allantoic fluid from chilled embryonated eggs is collected. Typically 8-10 ml of raw allantoic fluid is obtained from one egg.

Concentration and isolation of whole virus particles from the allantoic fluid

Clarification of the fluid

The obtained allantoic fluid is clarified by centrifugation at moderate speed (in the range of 4000 - 14000 g)

Precipitation step

For the virus clearing, a saturated ammonium sulfate solution is added so as to obtain a final ammonium sulfate concentration of 0.5 mol / L. After sedimentation for at least 1 hour, the precipitate is removed by filtration through depth filters (typically 0.5 μίτι).

Filtering

The clarified batch of whole virus particles is filtered through filter membranes ending with a certified sterile membrane (typically 0.2 μη).

Ultrafiltration

The sterile filtered crude stock of monovalent whole virus particles is concentrated at least 6-fold in the ΒΙΟΜΑΧ ™ 1000 kDa MWCO membrane cassettes. The concentrated retentate is washed with phosphate buffered saline at least 1.8 times. 14 PL 206 435 Β1

Centrifugation in a sucrose gradient

The influenza virus is concentrated by isopyclic centrifugation on a linear sucrose gradient (0.55% (w / v). The flow rate is 8-15 L / hour.

After completion of centrifugation, four different fractions are recovered from the contents of the centrifuge (sucrose is measured in a refractometer): - fraction 1 55-52% sucrose - fraction 2 about 52-38% sucrose - fraction 3 38-20% sucrose * - fraction 4 20 -0% sucrose * depending on the virus strain: fraction 3 can be reduced to 15% sucrose.

For the further production of the vaccine, only fraction 2 or fraction 2 can be used together with the additionally purified fraction 3.

Fraction 3 is washed in the diafiltration process with phosphate buffer to reduce the sucrose content to an indicative value of less than 6%. Alternatively, you can skip this stage. The influenza virus present in this dilution is centrifuged to remove soluble contaminants.

The sediment is resuspended and thoroughly mixed to obtain a homogeneous suspension. Fraction 2 and the resuspended fraction 3 are pooled and phosphate buffer is added to obtain a volume of about 40 liters. This product is a monovalent whole virus concentrate.

Sucrose gradient centrifugation with sodium deoxycholate

The monovalent whole influenza virus concentrate is loaded into an ENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrose gradient (0.55% (w / v)) over which an additional sodium deoxycholate gradient is overlaid. Tween 80 is present during cleavage at concentrations up to 0.1 (w / v) and for B strain viruses up to 0.5 mM tocopherol succinate is added. The maximum sodium deoxycholate concentration is 0.7-1.5% (w / v) and is strain dependent. The flow rate is 8-15 liters / hour.

After completion of the centrifugation, the rotor contents are recovered in three different fractions (the sucrose is measured in a refractometer). Fraction 2 is used for further processing. The sucrose limits (47-18%) vary from strain to strain and are set after evaluation.

Sterile filtration

The split virus fraction is filtered on filter membranes ending with a 0.2 μπι membrane. For dilution, a solution containing a phosphate buffer, Tween 80 at 0.025% (w / v) and (in the case of B strains) tocopherol succinate at a concentration of 0.5 mM is used. The final volume of the filtered fraction is 2-5 times the original volume of the fraction.

In activation

The filtered monovalent material is incubated at 22 ± 2 ° C for a maximum of 84 hours (depending on the virus strain, the incubation time may be reduced). Phosphate buffer containing 0.025% (w / v) Tween 80 is then added to reduce the total protein content to a maximum of 450 µg / ml. For B-strain viruses, phosphate buffered saline containing 0.025% (w / v) Tween 80 and 0.25 mM tocopherol succinate is used for dilution and reduction of total protein content to 450 µg / ml. Formaldehyde is added to a final concentration of 100 µg / ml and inactivation is carried out at 20 ± 2 ° C for at least 72 hours.

Ultrafiltration

The inactivated split viral material is concentrated at least two times in a ultrafiltration unit, equipped with cellulose acetate membranes with 20 kDa MWCO. The material is then washed with phosphate buffer containing 0.025% (w / v) Tween 80 followed by phosphate buffered saline containing 0.01% (w / v) Tween. For the B-strain virus, phosphate buffered saline containing 0.01% (w / v) Tween 8 and 0.1 mM tocopherol succinate is used for washing.

Final sterile filtration

The ultrafiltrated material is filtered through membranes ending with a 0.2 μίτι membrane. The filtration membranes are rinsed and the material is diluted if necessary so that the protein concentration does not exceed 500 μ9 / ηηη, using phosphate buffered saline containing 0.01% (w / v) Tween 80 and (for B viruses) 0.1 mM tocopherol succinate. Storage

The final monovalent batch is stored at 2 - 8 * 0 for a maximum of 18 months.

Stability

Table 4. Comparison of changes in HA content (pg / ml) over time, detected in the SRD assay in the final monovalent lots

Strain Stabilizer After production 4 weeks at 30 ° Ο 6 months at 2-8 ° CB / Johannesburg / 5/99 Tocopherol succinate 214 196 (92%) 206 (96%) B / Johannesburg / 5/99 ** None 169 107 ( 63%) 153 (90%) * prepared according to example 1 without tocopherol succinate

Example 5b. Influenza antigen preparation using α-tocopherol succinate as stabilizer in a thiomersal free vaccine

A preferred variation of the method described in Example 5a is described below.

After the whole virus particles were collected, they were precipitated (using ammonium sulfate). This is followed by a clarification step in which the liquid is clarified by a moderate speed centrifugation (range 4,000 - 14,000 g). Thus, the order of the precipitation and clarification steps is reversed compared to example 5a.

The sterile filtration, ultrafiltration and ultracentrifugation steps (sucrose gradient centrifugation) are performed as in Example 5a. However, there is no need to rework the fractions from the ultracentrifugation step.

The rest of the process is carried out as described in example 5a.

So, in short, the process in this example is as follows:

Collecting

Precipitation (ammonium sulfate)

Clarification

Sterile filtration

Ultrafiltration

Ultracentrifugation

Cleavage (preferably with sodium deoxycholate)

Sterile filtration

Inactivation

Ultrafiltration

Final sterile filtration

Another preferred embodiment of example 5a includes a preliminary filtration step prior to the first sterile filtration. For this purpose, a membrane is used which does not provide sterile filtration, but allows the removal of impurities, e.g. albumin, prior to sterile filtration. This can lead to better performance. A suitable prefiltration membrane has pores of about 0.8-1.8 μίτι, e.g. 1.2 μίτι. A preliminary filtration step can be used in the scheme of example 5a or example 5b.

Example 6: Preparation of an influenza vaccine using α-tocopherol succinate as a stabilizer in a thiomersal-free vaccine

The monovalent final batches of the three strains, A / New Caledonia / 20/99 (H1N1) IVR-116, A / Panama / 2007/99 (H3N2) Resvir-17 and B / Yamanashi / 166/98, were prepared as described in Example 5. Combination

Appropriate amounts of monovalent final batches were pooled to give a final concentration of 30 μ9 / ιτιΙ for A / New Caledonia / 20/99 (H1N1) IVR-116 and A / Panama / 2007/99 (H3N2) Resvir-17 and 36 μ9 / respectively. ιτιΙ for B / Johannesburg / 5/97. The concentrations of Tween 80 and Triton X -100 were set at 580 µ9 / ιτιΙ and 90 µg / ml, respectively. The final volume was made up to 3 L with phosphate buffered saline. The trivalent linkage was filtered last through a 0.8 μίτι cellulose acetate membrane and the final trivalent batch was obtained. A trivalent final batch was filled into syringes, each at least 0.5 ml.

Table 5. Comparison of changes in HA content over time, detected in the SRD test, in the final trivalent batches

Vaccine preparation Strain Month 0 4 weeks at 30 ^ 0 6 months at 2-8 ° C Influenza vaccine without stabilizer A / NCal / 20/99 31 32 30 A / Pan / 2007/99 31 34 33 B / Joh / 5/99 * 35 25 31 Influenza vaccine containing a-tocopherol succinate A / NCal / 20/99 34 35 34 A / Pan / 2007/99 33 33 34 B / Joh / 5/99 ** 29 25 28

Preparation with a target concentration of 39 pg / ml. ** Preparation with a target concentration of 34 pg / ml.

Example 7. Preparation of an influenza virus antigen preparation using sodium lauryl sulfate as a stabilizer in a vaccine without preservatives (with reduced thiomersal content)

The monovalent B / Johannesburg / 5/99 virus whole particle concentrate was obtained as described in Example 1.

Sucrose gradient centrifugation with sodium deoxycholate

The monovalent whole-particle concentrate of influenza virus is loaded into an ENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrose gradient (0.55% (w / v)) over which an additional sodium deoxycholate gradient is overlaid. Tween 80 is present at a concentration of up to 0.1% (w / v) during the splitting. The maximum sodium deoxycholate concentration is 0.7-1.5% (w / v) and depends on the strain. The flow rate is 8-15 l / hour.

After completion of the centrifugation, three different fractions were recovered from the rotor contents (sucrose is measured in a refractometer). Fraction 2 is used for further processing. The sucrose limits (47-18%) vary from strain to strain and are set after evaluation.

Sterile filtration

A 10 ml aliquot of fraction 2 was taken for further processing. The split virus fraction was filtered on filter membranes ending with a 0.2 μίτι membrane. Phosphate buffer containing 0.025% (w / v) Tween 80 and 0.5 mM sodium lauryl sulfate is used for dilution. The final volume of the filtered fraction is 2-5 times the original volume of the fraction.

In activation

The filtered monovalent material is incubated at 22 ± 2 ° C for a maximum of 84 hours (depending on the virus strain, the incubation time may be reduced). Phosphate buffered saline containing 0.025% (w / v) Tween 80 and 0.5 mM sodium lauryl sulfate are then added to reduce the total protein content to a maximum of 250 µg / ml. Formaldehyde is added to a final concentration of 50 µg / ml and inactivation occurs at 20 ± 2 ° C for at least 72 hours.

Ultrafiltration

The inactivated split viral material is concentrated at least two times in a ultrafiltration unit, equipped with cellulose acetate membranes with 20 kDa MWCO. The material is then washed with 4 volumes of phosphate buffered saline containing 0.01% (w / v) Tween and 0.5 mM sodium lauryl sulfate.

Final sterile filtration

The ultrafiltrated material is filtered through filter membranes ending with a 0.2 μηι membrane. The filtration membranes are washed and the material is diluted if necessary so that the protein concentration does not exceed 500 μg / ml, using phosphate buffered saline containing 0.01% (w / v) Tween 80 and 0, 5 mM sodium lauryl sulfate. 17 PL 206 435 Β1

Storage

The final monovalent batch is stored at 2 - δ'Ο.

Table 7. Comparison of changes in HA content over time, detected in the SRD test in the final monovalent batches

Stabilizer After 4 weeks preparation at 30 B / Johannesburg / 5/99 None * 182 139 (77%) B / Johannesburg / 5/99 Sodium lauryl sulfate 288 264 (92%) * prepared by the method of Example 7 without addition of lauryl sulfate.

Example 8. Preparation of an influenza virus antigen preparation using Plantacare or La-reth-9 as stabilizers in a vaccine without preservatives (with reduced thiomersal content)

The monovalent B / Yamanashi / 166/98 virus whole particle concentrate was prepared as described in Example 1.

Fragmentation

The monovalent influenza virus whole particle concentrate is diluted to a protein concentration of 1000 pg / ml with phosphate-buffered saline at pH 7.4. Plantacare® 2000 UP or Laureth-9 is added to a final concentration of 1% (w / v). The material is gently agitated for 30 minutes. The material is then layered onto a 15% (w / w) sucrose pad in the vessel. Ultracentrifugation in a Beckman SW 28 centrifuge is carried out for 2 hours at 25,000 rpm and 20 ° C.

Sterile filtration

The supernatant was processed further. The split virus fraction is filtered on filter membranes ending with a 0.2 μίτι membrane.

In activation

Phosphate-buffered saline is added when necessary to reduce the total protein content to a maximum of 500 µg / ml. Formaldehyde is added to a final concentration of 100 µg / ml and inactivation is carried out at 20 ± 2 ° C for at least 6 days.

Ultrafiltration

The concentrations of Tween 80 and Triton X 100 in the inactivated material are set at 0.15% and 0.02%, respectively. The inactivated split viral material is concentrated at least two times in a ultrafiltration unit, equipped with cellulose acetate membranes with 30 kDa MWCO. The material is then washed with 4 volumes of phosphate buffered saline.

Final sterile filtration

The ultrafiltrated material is filtered through membranes ending with a 0.2 μηι membrane. The filtration membranes are washed and the material is diluted so that the protein concentration does not exceed 500 µg / mL using phosphate buffered saline.

Storage

The final monovalent batch of virus is stored at 2-8 * 0.

Table 8. Comparison of changes in HA content over time, detected in the SRD test in the final monovalent batches

Stabilizer After production 4 weeks at 30 ° CB / Yamanashi / 166/98 - 143 98 (68%) B / Yamanashi / 166/98 Plantacare® 2000 UP 476 477 (100%) B / Yamanashi / 166/98 Laureth-9 468 494 (> 100%)

Example 9. Clinical studies of an α-tocopherol stabilized influenza vaccine (thiomersal reduced) in the elderly, intradermally and intramuscularly. Preparation of influenza virus antigen preparation

A monovalent split vaccine was prepared according to the following procedure. Preparation of viral inoculum On the day of inoculation with embryo eggs, fresh inoculum is prepared by mixing the working virus culture with phosphate buffered saline containing 0.5 mg / ml gentamicin sulfate and 25 μg / ml hydrocortisone . (depending on the viral strain). Virus inoculum is stored at 2-8 ° C.

Inoculation of eggs containing embryos

9-11 day old embryonated eggs are used for virus replication. The shells are disinfected. The eggs are inoculated with 0.2 ml of viral inoculum material. Inoculated eggs are incubated at an appropriate temperature (depending on the virus strain) for 48-96 hours. At the end of the incubation period, the embryos are killed by chilling and the eggs are stored for 12-60 hours at 2-8 ° C.

Collecting

Allantoic fluid from chilled embryonated eggs is collected. Typically 8-10 ml of raw allantoic fluid is obtained from one egg.

Concentration and isolation of whole virus particles from the allantoic fluid 1. Clarification of the fluid

The obtained allantoic fluid is clarified by centrifugation at moderate speed (in the range of 4000 - 14000 g). 2. The adsorption stage

To obtain a CaHP04 gel in the clear virus stock, 0.5 mol / L Na2HP04 and 0.5 mol / L CaCl2 solution are added so as to obtain a final CaHP04 concentration of 1.5 g - 3.5 g CaH-PO4 / liter, depending on the strain of virus.

After sedimentation for at least 8 hours, the supernatant is removed and the influenza virus pellet is redissolved by adding 0.26 mol / l EDTA-Na2 solution depending on the amount of CaHP04 used. 3. Filtration

The resuspended precipitate is filtered through a 6 µΐ filtration membrane. 4. Sucrose gradient centrifugation

The influenza virus is concentrated by isopyclic centrifugation on a linear sucrose gradient (0.55% (w / v)) containing 100 µg / ml Thiomersal. The flow rate is 8-15 l / hour.

At the end of centrifugation, four different fractions are recovered from the rotor (sucrose is measured in a refractometer): - fraction 1 55-52% sucrose - fraction 2 about 52-38% sucrose - fraction 3 38-20% sucrose * - fraction 4 20-0 % sucrose * depending on the virus strain: fraction 3 can be reduced to 15% sucrose.

Only fractions 2 and 3 are used for the further production of the vaccine.

Fraction 3 is washed in the diafiltration process with phosphate buffer to reduce the sucrose content to less than about 6%. The influenza virus present in this dilution is centrifuged to remove soluble contaminants.

The sediment is resuspended and thoroughly mixed to obtain a homogeneous suspension. Fraction 2 and the resuspended fraction 3 are combined and phosphate buffer added to obtain a volume of approximately 40 liters, a volume approximately 120,000 eggs / lot. This product is the monovalent whole virus particle concentrate. 5. Sucrose gradient centrifugation with sodium deoxycholate

The monovalent whole influenza virus concentrate is loaded into an ENI-Mark II ultracentrifuge. The K3 rotor contains a linear sucrose gradient (0.55% (w / v)) over which an additional sodium deoxycholate gradient is overlaid. Tween 80 is present at a concentration of up to 0.1 (w / v) during the splitting and tocopherol succinate is added in concentrations of up to 0.5 mM for B-strain viruses. The maximum sodium deoxycholate concentration is 0.7-1.5% (w / v) and is strain dependent. The flow rate is 8-15 liters / hour.

After completion of the centrifugation, the rotor contents are recovered in three different fractions (the sucrose is measured in a refractometer). Fraction 2 is used for further processing. The sucrose limits (47-18%) vary from strain to strain and are set after evaluation. 6. Sterile filtration

The split virus fraction is filtered on filter membranes ending with a 0.2 μίτι membrane. Phosphate buffer containing 0.025% Tween 80 (w / v) and (for B virus strains) 0.5 mM tocopherol succinate is used for dilution. The final volume of the filtered fraction is 2-5 times the original volume of the fraction. 7. In activation

The filtered monovalent material is incubated at 22 ± 2 ° C for a maximum of 84 hours (depending on the virus strain, the incubation time may be shortened). Phosphate buffer containing 0.025% (w / v) Tween 80 is then added to reduce the total protein content to a maximum of 250 µg / ml. For B-strain viruses, phosphate buffered saline containing 0.025% (w / v) Tween 80 and 0.25 mM tocopherol succinate is used to dilute the total protein content to 250 µg / ml. Formaldehyde is added to a final concentration of 50 µg / ml and inactivation is carried out at 20 ± 2 ° C for at least 72 hours. 8. Ultrafiltration

The inactivated split viral material is concentrated at least two times in a ultrafiltration unit, equipped with cellulose acetate membranes with 20 kDa MWCO. The material is then washed with phosphate buffer containing 0.025% (w / v) Tween 80 followed by phosphate buffered saline containing 0.01% (w / v) Tween. For B-strain virus, phosphate buffered saline containing 0.01% (w / v) Tween 80 and 0.1 mM tocopherol succinate is used for washing. 9. Final sterile filtration

The ultrafiltrated material is filtered through membranes ending with a 0.2 μίτι membrane. The filter membranes are washed and the material is diluted if necessary so that the protein concentration does not exceed 1000 μg / ml but the hemagglutinin concentration exceeds 180 μg / ml, using phosphate buffered saline containing 0.01% (wt. / vol) Tween 80 and (for B viruses) 0.1 mM tocopherol succinate. 10. Storage

The final monovalent virus batch is stored at 2-8 ° C for a maximum of 18 months. B Manufacture of an influenza vaccine

The final monovalent batches of the three strains, A / New Caledonia / 20/99 (H1N1) IVR-116, A / Panama / 2007/99 (H3N2) Resvir-17 and B / Johannesburg / 5/99, were prepared as described in section A above Connecting

Appropriate amounts of monovalent final batches were combined to reach a final concentration of 60 Mg / ml of A / New Caledonia / 20/99 (H1N1) IVR-116 and A / Panama / 2007/99 (H3N2) Resvir-17 and 68 pg / ml respectively. for B / Johannesburg / 5/99. The concentrations of Tween 80, Triton X-100 and tocopherol succinate were set at 1000 pg / ml, 110 pg / ml and 160 µg / ml, respectively. The final volume was made up to 3 liters with phosphate buffered saline. The trivalent linkage was filtered through a membrane terminating with a 0.8 μίτι cellulose acetate membrane and the final trivalent lot was obtained. The trivalent final lot was placed in syringes, each with at least 0.165 ml.

Administration of the vaccine

The vaccine was delivered in pre-filled syringes and was administered intradermally into the deltoid region. The intradermal injection needle (ID) described in EP1092444 was used, with a skin penetration restrictor to ensure correct intradermal injection. Since the formation of a blister (clump) at the injection site is a sign of good quality ID administration, an experienced researcher measured the exact bladder size 30 minutes after each vaccination.

One dose (100 μΙ) contained the following ingredients:

Haemagglutinin from three influenza strains Al New Caledonia / 20/99 6.0 pg A / Panama / 2007/99 6.0 pg B / Johannesburg 5/99 6.0 pg Thiomersal preservative 0.4 pg - 0.8 pg 20 PL 206,435 Β1 B The above vaccine was compared to a conventional trivalent split influenza vaccine: Fluarix ™. Fluarix was supplied in pre-filled syringes and was administered intramuscularly into the deltoid muscle. A needle at least 2.5 cm (1 inch) long (23 gauge) was used to ensure correct intramuscular administration.

One dose (0.5 ml) contained the following ingredients:

Haemagglutinin from three influenza strains A / New Caledonia / 20/99 15.0 Mg A / Panama / 2007/99 15.0 Mg B / Johannesburg 5/99 15.0 Mg Preservative thiomersal 50 Mg

Results The mean age of the entire study group at the time of vaccine administration was 70.4 ± 6.2 years, standard deviation (S, D), the ratio of women to men was 1.7: 1.

Immunogenicity Results: Variable Derivative Analysis of Immunogenicity: Variable Influenza Variable, ID (N = 65) FluarixTM, IM (N = 65) 1 2 3 4 5 6 7 8 GMT GMT LL UL GMT LL UL A / New Caledonia before 99, 5 76.9 128.7 90.0 70.1 115.7 165.1 each 129.2 211.0 174.3 133.3 227.9 A / Panama before 75.5 54.7 104.2 69, 2 51.9 92.4 after 128.6 99.1 166.8 164.3 126.0 214.1 B / Johannesburg before 236.0 187.7 296.8 222.6 176.9 280.2 after 341 , 2 276.0 421.7 402.4 312.1 518.9 Percentage of serocon versions% LL UL% LL UL A / New Caledonia 15.4 7.6 26.5 18.5 9.9 30.0 A / Panama 20.0 11.1 31.8 29.2 18.6 41.8 B / Johannesburg 9.2 3.5 19.0 16.9 8.8 28.3 Conversion Factor GMR LL UL GMR LL UL A / New Caledonia 1.7 1.4 2.0 1.9 1.6 2.3 A / Panama 1.7 1.4 2.1 2.4 1.9 3.0 B / Johannesburg 1.4 1.2 1.7 1.8 1.5 2.1 Rate of serological protection% LL UL% LL UL A / New Caledonia before 87.7 77.2 94.5 90.8 81.0 96.5 after 92.3 83, 0 97.5 96.9 89.3 99.6 A / Panama before 75.4 63.1 85.2 81.5 70.0 90.1 21 PL 206 435 Β1 cont. table 1 2 3 4 5 6 7 8 after 90.8 81.0 96.5 93.8 85.0 98.3 B / Johannesburg before 98.5 91.7 100.0 96.9 89.3 99.6 100.0 94.5 100.0 98.5 91.7 100.0 N: number of patients with available results;%: percentage of patients with a given parameter; LL / UL: lower and upper limit of 95% Cl; Before: at the time of vaccination; After: 21 days post vaccine injection Pain at the injection site was reported in 10/65 (15.4%) of the vaccinated subjects, and was the most common symptom following intramuscular administration of Fluarix ™. In the intradermal vaccination group, pain was reported in 3/65 (4.6%) vaccinated persons. This difference was statistically significant (p = 0.038; Fisher's exact test). Therefore, it is preferable to administer the product intradermally with a reduced thiomersal content.

Conclusions

The intradermal and intramuscular administration of the thimerosal-reduced vaccine in the elderly population can provide 100% serological protection.

Comparable response to vaccination in terms of geometric mean titers, percent seroconversion, seroconversion rates and conversion rates was achieved in vaccinated subjects after both intradermal and intramuscular administration, with ID patients receiving 2.5 times less antigen. There were no discernible differences between the two treatment groups in the overall incidence of vaccine-related systemic symptoms observed / tested.

Example 10. Intradermal administration of a thiomersal reduced vaccine

The immunogenicity of a thiomersal-reduced split influenza vaccine prepared as described in Example 9 (except that pooling was done independently and syringes were not filled with vaccine) was assessed after intradermal administration to guinea pigs using a standard needle.

Groups of 5 animals each initially receive intranasal starting dose of all inactivated trivalent influenza virus containing 5 μίτι each HA in a total volume of 200 μΙ. After 28 days, the animals were vaccinated intradermally or intramuscularly. Intradermal doses containing 0.1, 0.3, or 1.0 µg thiomersal-reduced trivalent split influenza virus in a volume of 0.1 ml were administered into the back of guinea pigs using a standard needle. An intramuscular dose containing 1.0 µg of trivalent split influenza virus with thiomersal reduced in a volume of 0.1 ml was administered to the hind leg of the guinea pig. The following groups were included in the study:

Group 1 - 0.1 µg trivalent split influenza virus with thiomersal ID reduced content;

Group 2 - 0.3 μηπ of trivalent split influenza virus with thiomersal ID reduced content;

Group 3 -1.0 pg trivalent split influenza virus with thiomersal ID reduced content;

Group 4 - 1.0 pg trivalent split influenza virus with thiomersal reduced IM content. The animals were blood drawn 14 days after vaccination and the titers of vaccination-induced antibodies were assessed using the known haemagglutinin inhibition (HI) test. The results are shown in Figure 1. Vaccination induced a strong HI response from all three strains. The observed lack of a clear dose-response relationship suggests that very low doses of the thiomersal-reduced antigen may still induce a strong HI antibody response after ID administration. There were no significant differences between the HI titers induced by IM and ID vaccination. Thus, the results obtained in guinea pigs confirm that thiomersal-reduced trivalent split influenza virus antigens induce similar levels of HI antibodies in animals after ID administration compared to IM administration. 22 PL 206 435 Β1

Example 11. Intradermal administration of a thiomersal reduced adjuvant vaccine

Protocol On day 0, guinea pigs were dosed at a starting dose of 5 µg of total inactivated trivalent influenza virus at 200 µΙ, intranasally.

Vaccination - Day 28 - A vaccine containing 0.1 µg HA per trivalent split influenza virus strain was prepared as described in Example 9 (except that the pooling step resulted in a final concentration of each antigen of 1.0 µg / ml, giving a dose of 0 1 µg in 100 µl compared to 60 µg / ml in example 9). The final trivalent adjuvant or non-adjuvant formulation was administered intradermally using 100 µL tuberculin syringes.

Blood Collection - Day 42

The adjuvant effect was assessed by measuring the antibody response by HI test (day 0.28.42).

All ID experiments were performed using an ordinary needle.

The G1-G5 results are for 5 groups of guinea pigs, 5 animals per group. G1 Split trivalent with reduced thiomersal 0.1 pg G2 Split trivalent with reduced thiomersal 0.1 pg + 3D-MPL 50 pg G3 Split trivalent with reduced thiomersal 0.1 pg + 3D-MPL 10 pg G4 Split trivalent with reduced thiomersal 0.1 pg + 3D-MPLin 50 pg + QS21 50 pg G5 Split trivalent thiomersal reduced 0.1 pg + 3D-MPLin 10 pg + QS21 10 pg 3D-MPLin + QS21 is an adjuvanted formulation that contains a monolayer vesicle cholesterol-containing, dioleoylphosphatidylcholine-containing lipid bilayer, wherein QS21 and 3D-MPL are bound to or embedded in the lipid bilayer. Such adjuvanted compositions are described in EP 0822831 B.

Anti-A / New Caledonia HI titers / 20/99 NC Before vaccination Before booster After booster G1 5 10 92 G2 5 10 70 G3 5 11 121 G4 7 9 368 G5 5 10 243

Anti-A HI titers / Panama / 2007/99 P Before vaccination Before booster dose After booster dose G1 5 485 7760 G2 5 279 7760 G3 5 485 8914 G4 7 485 47051 G5 5 320 17 829 23 PL 206 435 Β1

Anti-B HI titers / Johannesburg / 5/99 J Before vaccination Before booster dose After booster dose G1 5 23 184 G2 5 11 121 G3 5 11 70 G4 6 15 557 G5 5 13 320

Thus, both the adjuvant and the non-adjuvant split thiomersal-reduced trivalent influenza virus antigen is a potent immunogen and is capable of eliciting a strong HI response when administered with both ID and IM. These responses appear to be at least as strong as those induced by typical Fluarix preparation.

Example 12. Comparison of thiomersal-free and thiomersal-free vaccines administered intradermally to pigs

Pigs that received the initial dose by the intradermal route were used as a model to evaluate the immunogenicity of the split influenza vaccine (with and without thiomersal). Since the vast majority of the population has had flu at least once, the flu vaccine must be able to stimulate an existing immune response. For this reason, the animals received a starting dose of virus antigens to best mimic the situation found in humans. In this study, 4 week old pigs received a starting dose of influenza virus antigens by the intranasal route. Six groups of five animals each received a starting dose of virus as follows:

Group 1 - Two starting doses of total trivalent inactivated virus (50 µg of each HA) on days 0 and 14; Group 2 - two starting doses of total trivalent inactivated virus (50 μς of each HA) on days 0 and 14; Group 3 - Single starting dose of all trivalent inactivated virus (50 µg of each HA) at day 0; Group 4 - two starting doses of total trivalent inactivated virus (25 μς of each HA) on days 0 and 14; Group 5 - Single starting dose of all trivalent inactivated virus (25 µg of each HA) at day 0; Group 6 - Two starting doses of whole trivalent inactivated virus (12.5 μg each HA) on days 0 and 14. On day 28 after the last starting dose, animals were vaccinated with 3 μg of each HA of the trivalent split antigen (strains A / New Caledonia H1N1, A / Panama H3N2 and B / Johannesburg) at 100 μΙ, by the intradermal route. Group 1 received a standard dose of Fluarix ™ containing thiomersal as preservative as the vaccine antigen. All other groups received the antigen without preservatives.

The HI results obtained in this experiment are shown in Figure 2 (anti-influenza anti-hemagglutination antibody titers in pigs that received different doses of antigen initially and were vaccinated with 3 micrograms of trivalent influenza antigen, with and without Thiomersal, by the intradermal route). In the present experiment, relatively low HI titers were induced over the B strain and the starting HI titers over the A / H3N2 strain are high. A beneficial effect is observed in terms of the response to vaccination when the starting dose of the antigen is reduced. In almost all cases, a reduction in the antigen concentration or the number of pre-doses (compared to the two 50 µg doses) resulted in an enhanced response to vaccination. While the response to vaccination of animals in Groups 1 and 2 that received twice the 50 μg starting dose is not very apparent, the preservative-free antigen (Group 2) appears to be at least as effective as Fluarix ™ (Group 1) under these conditions. A strong response to vaccination with the intradermal route of trivalent influenza virus antigen, devoid of preservatives, is clearly discernible to animals given different starting doses (Groups 3-6). This response is discernible even for the B strain, although HI titers remain low. 24 PL 206 435 Β1

Claims 1. An inactivated influenza virus preparation containing a haemagglutinin antigen stabilized without the use of thiomersal or at low levels of thiomersal, the haemagglutinin being detected by the SRD assay, characterized in that the preparation contains sufficient α-tocopherol succinate to stabilize the haemagglutinin. 2. An inactivated influenza virus preparation according to claim 1 The method of claim 1, characterized in that it contains α-tocopherol succinate in a concentration of 1 µg / ml -10 mg / ml. 3. An inactivated influenza virus preparation according to claim 3; The method of claim 2, characterized in that it contains α-tocopherol succinate in a concentration of 10-500 ng / ml. 4. An inactivated influenza virus preparation according to claim 1 The method according to any of the preceding claims, wherein the influenza virus antigen preparation is selected from the group consisting of split virus antigen preparations, subunit antigens, and chemically inactivated or ultraviolet or heat inactivated whole virus. 5. An inactivated influenza virus preparation according to claim 5 The method of claim 4, wherein the influenza antigen preparation is a split virus antigen preparation. 6. An inactivated influenza virus preparation according to claim 6 6. An inactivated influenza virus preparation according to claims 1 to 5, characterized in that it contains both A and B strain he-maglutinin. The process of claim 6 which is a trivalent virus preparation. 8. An inactivated influenza virus preparation according to claim 1 Influenza vaccine, characterized in that it comprises an influenza virus preparation as defined in claims 1 to 7, characterized in that it contains stabilized haemagglutinin of influenza B strain. 10. Influenza vaccine according to claims 1-8. The method of claim 9, wherein the concentration of the haemagglutinin antigen for each influenza strain is 1 - 1000 pg / ml as determined by the SRD test. 11. The influenza vaccine according to claim 11 The pharmaceutical composition according to claim 9 or 10, further comprising an adjuvant. 12. A method for producing a stable haemagglutinin antigen, characterized by purifying the antigen in the presence of α-tocopherol or a derivative thereof, such as α-tocopherol succinate. 13. Use of an inactivated influenza virus preparation for the production of a vaccine as defined in claim 1, 9-11, for prophylaxis of infection or illness of influenza in a subject. 14. Use according to claim 1 The method of claim 13, wherein the vaccine is administered via the intradermal intranasal intramuscular route, orally or subcutaneously. 15. Use of α-tocopherol succinate as a haemagglutinin stabilizer in the manufacture of an influenza vaccine.